Copy Number Ratio Research Articles

Concentration and reduction efficiency of vancomycin-resistant heterotrophic bacteria and vanA and vanB genes in wastewater treatment unit processes

ObjectivesThis study elucidated the distribution and fate of vancomycin (VCM)-resistant heterotrophic bacteria (HTB) and resistance genes, vanA and vanB, during each treatment unit process of a wastewater treatment plant (WWTP). MethodsSeveral bacterial counts as well as copy numbers of vanA and vanB genes were determined in each wastewater and sludge sample. In addition, HTB strains isolated from wastewater and sludge were analyzed for VCM susceptibility. Then, the fate and reduction ratios of each bacterial count, copy number of vanA and vanB genes, and the existence ratio of VCM-resistant HTB strains in the wastewater treatment unit process were evaluated. ResultsVCM-resistant HTB were detected in all wastewater and sludge samples, and their existence ratio decreased along the treatment process (92.9% in influent wastewater to 39.4% in chlorinated water). Notably, most of the HTB isolated from the influent wastewater were resistant to 8.0 µg/mL of VCM, strongly suggesting that a significant number of antibiotic-resistant bacteria are flowing into the WWTP from urban areas through the sewage system. The vanA and vanB genes were also detected in all wastewater and sludge, with high copy numbers (102-104 copies/mL) even in chlorinated water samples. ConclusionsResults revealed that residual VCM-resistant HTB, and resistance genes, which could not be completely removed, were ubiquitously released into the aquatic environment. Furthermore, a high existence ratio of VCM-resistant HTB and high copy numbers of resistance genes were also detected in the sludge, indicating that they are constantly circulating in the WWTP via the returned sludge.

Digital PCR-Based Characterization of a g10evo-epsps Gene-Specific Matrix Reference Material for Its Food and Feed Detection.

g10evo-epsps is a novel glyphosate herbicide-resistant gene that has been transferred to various crops such as soybean, corn, cotton, and rice. Here, we developed a gene-specific digital Polymerase Chain Reaction (dPCR) detection method for absolute quantitative analysis of g10evo-epsps, and characterized g10evo-epsps certified reference materials (CRM) using ZUTS-33 soybean powder as the candidate material. Stability tests of matrix CRMs demonstrate that these CRMs can be stored stably for 6 months and transported for 10 days at room temperature and withstand summer high temperatures (below 60 °C). CRM characterization is based on the copy number ratio of g10evo-epsps to lectin. Eight qualified laboratories independently validated the CRM using dPCR method, with a measurement of 0.98 (copy/copy) and an extended uncertainty of 0.08 (copy/copy). The g10evo-epsps matrix CRM described here may be used for qualitative and quantitative testing, method evaluation, laboratory quality control, and other related fields.

ChosenHRDw: A novel tool for the detection of homologous recombination deficiency(HRD) using low-pass whole-genome sequencing.

e17573 Background: Homologous recombination deficiency(HRD) as a promising biomarker holds predictive and prognostic value in anticancer therapies, especially in ovarian cancer. Although next-generation sequencing techniques such as whole-exome sequencing (WES) and targeted sequencing have proven to be powerful detection of HRD, the latest low-pass (1x) whole-genome sequencing (WGS) with characteristic large-scale patterns for HRD detection status, as a cost-effective screening strategy, have established feasibility. In this study, we developed the ChosenHRDw, a comprehensive algorithm, which utilizing low-pass WGS to effectively classify the HRD status by defining the high or low HRD score. Methods: In this work, a correction method base on GC-content was applied. Then we took a window size of 1Mb resolution, and removed the error alignments genome bins basing on the healthy cohort data. Baseline of each bins was constructed from the data of 100 healthy volunteers. We characterized and quantified 8 HRD-related signatures: LOH score, TAI score, LST score, chromosomal instability index(CIN index), wGII(weighted genome instability index), CNV ratio (copy number ratio in bins), HRR index (HRR gene instability index), TFBS ratio (copy number ratio in transcription factor binding sites) as the variables in a random forest model. Results: A total of 140 patients (pts) were enrolled in the discovery cohort, which including 70 high HRD score and 70 low HRD pts were confirmed by HRDetect on paired tumor/normal samples via 1123plus-genes panel targeted sequencing. The AUC was 0.93. Independent validation was conducted in a validation cohort of 60 pts, which HRD high or low pts were 30 and 30, respectively. The sensitivity = 0.87 and specificity = 0.9. Conclusions: We classified HRD into high vs low using ChosenHRDw, which showing strong concordance. Our analyses demonstrated that the prediction of HRD status can be achieved from low-pass WGS. Due to the limitation of relatively small sample size, real-world studies with a larger number of patients are needed to verify our algorithm in the future.

Single and multi-laboratory validation of a droplet digital PCR method

The authorisation of genetically modified food and feed in the EU is subject to the provision of evidence of safety and of the availability of reliable analytical methods. These methods represent an essential tool for official laboratories to enforce a harmonised market control.Here the validation of droplet digital PCR (dPCR) methods has been performed for studying if the performance and acceptance parameters set by EU and other international guidelines for the analysis of genetically modified organisms (GMO) in food and feed are suitable and achievable also with such methods. The single-laboratory validation study showed that performance requirements set for GMO analysis by real time PCR can also be used to assess dPCR-based methods. Moreover, trueness and precision were assessed for both simplex and duplex formats in a multi-laboratory validation study organised according to international standards. Overall, the data on trueness, repeatability and reproducibility precision resulting from the collaborative study are satisfying the acceptance criteria for the respective parameters as stipulated in the EU and other international guidance such as the Codex Committee on Methods of Analysis and Sampling (CCMAS). For instance, the duplex droplet dPCR method for MON810 showed relative repeatability standard deviations from 1.8% to 15.7%, while the relative reproducibility standard deviation was found to be between 2.1% and 16.5% over the dynamic range studied. Moreover, the relative bias of the dPCR methods was well below 25% across the entire dynamic range.In addition, other aspects supporting the application of digital PCR for the control of GMOs on the market were experimentally assessed such as the conversion of the measurement results from copy number ratio to mass fraction, the influence of the DNA extraction step and of the ingredient content. It was found that the DNA extraction step added only a limited contribution to the variability of the measurement results under the studied conditions. The decreasing amount of the target ingredient content may decrease the level of precision of the method, although within the acceptance range of GMO performance parameters.

Development of metrological support for nucleic acid measurements

Measurements of nucleic acid have become an essential part of analytical applications in many fields including life science, medicine, veterinary medicine and biotechnology. Peculiarities of these measurements are largely determined by the uniqueness of quantity and analyte definitions. Quantitative PCR, applied for routine nucleic acids measurements, is a relative method demanding external calibrators with quantity values traceable to corresponding reference materials. Direct nucleic acid measurements became possible after introduction of the digital PCR (dPCR) method. Ensuring accurate, comparable and traceable to SI nucleic acids measurements is the focus of Nucleic Acid Analysis Working Group of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology. A number of pilot and key comparisons carried out by Working Group have shown that application of dPCR allows participating National Metrological Institutes to obtain comparable results of measurements of the sequence copy number concentrations and copy number ratios.

The spectrum of HER2 expression in breast cancer: linking immunohistochemistry quantification with in situ hybridization assay.

We aimed to document the pathological characteristics of breast cancer (BC) cases with different scores of HER2 by immunohistochemistry (IHC), as well as to establish a relationship between HER2 expression and HER2 amplification by in situ hybridization (ISH). A cohort of 258 primary BC cases was evaluated for HER2 gene amplification with bright-field ISH. All HER2-negative and HER2-positive cases by IHC were concordant with the ISH classification. BC cases with score of 0 had lower average of HER2 copy number compared to cases with score of 1 + . HER2-equivocal cases by IHC had intermediate pathological characteristics between HER2-negative and HER2-positive cases. About 12% of HER2-equivocal cases were classified as ISH-positive. HER2-equivocal cases with HER2 gene amplification had proliferation index, HER2/CEP17 ratio, and average of HER2 copy number between HER2-equivocal cases without HER2 gene amplification and HER2-positive cases by IHC. Additionally, HER2-equivocal cases with HER2 amplification had score of 2 + in at least 50% of the total tumor area, with a proportion of ISH-positive cases increasing with the amount of score of 2 + present in the tumor. The quantification of score of 2 + in the tumor predicted the ISH classification with an AUC of 0.902. A logistic regression model using the same HER2 quantification and the nuclear score was able to increase the abovementioned prediction to an AUC of 0.929. As such, we were able to link HER2 quantification by IHC and morphological analysis with HER2 amplification by ISH.

Metabolic Reprogramming in Response to Alterations of Mitochondrial DNA and Mitochondrial Dysfunction in Gastric Adenocarcinoma

We used gastric cancer cell line AGS and clinical samples to investigate the roles of mitochondrial DNA (mtDNA) alterations and mitochondrial respiratory dysfunction in gastric adenocarcinoma (GAC). A total of 131 clinical samples, including 17 normal gastric mucosa (N-GM) from overweight patients who had received sleeve gastrectomy and 57 paired non-cancerous gastric mucosae (NC-GM) and GAC from GAC patients who had undergone partial/subtotal/total gastrectomy, were recruited to examine the copy number and D310 sequences of mtDNA. The gastric cancer cell line AGS was used with knockdown (KD) mitochondrial transcription factor A (TFAM) to achieve mitochondrial dysfunction through a decrease of mtDNA copy number. Parental (PT), null-target (NT), and TFAM-KD-(A/B/C) represented the parental, control, and TFAM knocked-down AGS cells, respectively. These cells were used to compare the parameters reflecting mitochondrial biogenesis, glycolysis, and cell migration activity. The median mtDNA copy numbers of 17 N-GM, 57 NC-GM, and 57 GAC were 0.058, 0.055, and 0.045, respectively. The trend of decrease was significant (p = 0.030). In addition, GAC had a lower mean mtDNA copy number of 0.055 as compared with the paired NC-GM of 0.078 (p < 0.001). The mean mtDNA copy number ratio (mtDNA copy number of GAC/mtDNA copy number of paired NC-GM) was 0.891. A total of 35 (61.4%) GAC samples had an mtDNA copy number ratio ≤0.804 (p = 0.017) and 27 (47.4%) harbored a D310 mutation (p = 0.047), and these patients had shorter survival time and poorer prognosis. After effective knockdown of TFAM, TFAM-KD-B/C cells expressed higher levels of hexokinase II (HK-II) and v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT, but lower levels of phosphorylated pyruvate dehydrogenase (p-PDH) than did the NT/PT AGS cells. Except for a higher level of p-PDH, the expression levels of these proteins remained unchanged in TFAM-KD-A, which had a mild knockdown of TFAM. Compared to those of NT, TFAM-KD-C had not only a lower mtDNA copy number (p = 0.050), but also lower oxygen consumption rates (OCR), including basal respiration (OCRBR), ATP-coupled respiration (OCRATP), reserve capacity (OCRRC), and proton leak (OCRPL)(all with p = 0.050). In contrast, TFAM-KD-C expressed a higher extracellular acidification rate (ECAR)/OCRBR ratio (p = 0.050) and a faster wound healing migration at 6, 12, and 18 h, respectively (all with p = 0.050). Beyond a threshold, the decrease in mtDNA copy number, the mtDNA D310 mutation, and mitochondrial dysfunction were involved in the carcinogenesis and progression of GACs. Activation of PDH might be considered as compensation for the mitochondrial dysfunction in response to glucose metabolic reprogramming or to adjust mitochondrial plasticity in GAC.

CNViz: An R/Shiny Application for Interactive Copy Number Variant Visualization in Cancer

Copy number variants (CNVs) comprise a class of mutation which includes deletion, duplication, or amplification events that range in size from smaller than a single-gene or exon, to the size of a full chromosome. These changes can affect gene expression levels and are thus implicated in disease, including cancer. Although a variety of tools and methodologies exist to detect CNVs using data from massively parallel sequencing (also referred to as next-generation sequencing), it can be difficult to appreciate the copy number profile in a list format or as a static image. CNViz is a freely accessible R/Bioconductor package that launches an interactive R/Shiny visualization tool to facilitate review of copy number data. As inputs, it requires genomic locations and corresponding copy number ratios for probe, gene, and/or segment-level data. If supplied, loss of heterozygosity (LOH), focal variant data [single nucleotide variants (SNVs) and small insertions and deletions (indels)], and metadata (e.g., specimen purity and ploidy) can also be incorporated into the visualization. The CNViz R/Bioconductor package is an easy-to-use tool built with the intent of encouraging visualization and exploration of copy number variation. CNViz can be used in a clinical setting as well as for research to study patterns in human cancers more broadly. The intuitive interface allows users to visualize the copy number profile of a specimen, dynamically change resolution to explore gene and probe-level copy number changes, and simultaneously integrate LOH, SNV, and indel findings. CNViz is available for download as an R package via Bioconductor. An example of the application is available at rebeccagreenblatt.shinyapps.io/cnviz_example.

A quantitative detection of mung bean in chestnut paste using duplex digital PCR

Highly manufacturing process of chestnut paste leaves a considerable space for Economically Motivated Adulteration (EMA) with cheaper ingredients such as mung bean. In this paper a novel quantitative detection of mung bean in chestnut paste using duplex digital PCR was reported. Two sets of primers and probes were designed according to mung bean and chestnut specific genomic genes suitable for duplex droplet digital PCR (ddPCR) and duplex chip digital PCR (cdPCR) to set up a mass ratio quantitative detection method for mung bean, a common alternative plant-derived ingredient in chestnut paste products. The manufacturing process of chestnut paste products was considered to establish the linear relationship formula between mass ratio and gene copy number (CN) ratio of the two ingredients. The limits of quantification for gene CN concentrations (LOQcopy) of mung bean and chestnut were both 6 copies/μL, at the same time a mass ratio of mung bean in chestnut paste range from 5% to 80% was able to be quantified accurately to provide technical support for the identification of fraudulent substitution or adventitious contamination.

A new strategy for distinguishing menstrual blood from peripheral blood by the miR-451a/miR-21-5p ratio

Distinction between menstrual blood and peripheral blood is vital for forensic casework, as it could provide strong evidence to figure out the nature of some criminal cases. However, to date no single blood-specific gene, including the most variable microRNAs (miRNAs) could work well in identification of blood source. In this study, we developed a new strategy for identification of human blood samples by using the copy number ratios of miR-451a to miR-21–5p based on 133 samples, including 56 menstrual blood and 47 peripheral blood, as well as 30 non-blood samples of saliva (10), semen (10) and vaginal secretion (10). The cut-off value and efficacy of the identification strategy were determined through receiver operating characteristic (ROC) analysis. Our results showed that when the miR-451a/miR-21–5p ratio below 0.929, the sample should be non-blood. In contrast, when the miR-451a/miR-21–5p ratio above 0.929 and below 10.201, the sample should be menstrual blood; and when this ratio above 10.201, the sample should be peripheral blood. External validation using 86 samples (62 menstrual blood and 24 peripheral blood samples) fully supported this strategy with the 100% sensitivity and 100% specificity. We confirmed that this result accuracy was not affected by various potential confounding factors of samples and different experimental platforms. We showed that 0.2 ng of total RNA from menstrual blood and peripheral blood was sufficient for qPCR quantification. In conclusion, our results provide an accurate reference to distinguish menstrual blood from peripheral blood for forensic authentication.

Real-time digital polymerase chain reaction (PCR) as a novel technology improves limit of detection for rare allele assays.

BackgroundTumor heterogeneity may lead to false negative test results for tissue biopsy-based companion diagnostic tests. Real-time polymerase chain reaction (PCR) and digital PCR assays are used to detect rare alleles in cell-free circulating DNA for liquid biopsies; however, those tests lack strong sensitivity at low allele frequencies. We show here a novel real-time digital PCR instrument that utilizes cycle-based amplification curves to further improve the sensitivity and quantification accuracy of digital PCR.MethodsThe novel real-time digital PCR instrument was compared to an endpoint digital PCR system to determine the sensitivity and quantification accuracy of both instruments. Samples were all thermal cycled on the real-time digital PCR instrument but were analyzed on both endpoint and real-time digital PCR instruments to compare the performance without introducing other variables. Contrived samples for epidermal growth factor receptor (EGFR) exon 19 deletion, T790M, and L858R point mutations as well as human epidermal growth factor receptor 2 (HER2) amplification were tested. Different mutant allele frequencies and wildtype to mutant gene copy number ratios were tested for EGFR and HER2, respectively.ResultsBy removing false positive datapoints using real-time amplification curves, real-time digital PCR improved sensitivity by lowering the baseline for wildtype samples. For EGFR 19del assay, samples with 2 or more fluorescein amidite (FAM) labeled positive wells are determined positive by real-time digital PCR, while a minimum of 5 FAM positive datapoints is needed by endpoint digital PCR. Improved limit of detection for EGFR 19del mutation was also observed. Real-time digital PCR also had better quantification accuracy and sensitivity, resulting in the mutant allele frequencies being closer to the expected values for all EGFR mutations, especially at very low allele frequencies. However, at high allele frequencies or for gene amplification assays, real-time digital PCR is comparable with endpoint digital PCR.ConclusionsThis novel technology with improved sensitivity is important and needed because it addresses current issues with liquid biopsy tests. Due to limited amounts of circulating tumor DNA (ctDNA) obtained for liquid biopsy tests, few copies of mutant alleles are expected. With the lower baseline of real-time digital PCR, false negative test results from tissue biopsy would be more effectively reduced, leading to more patients receiving the targeted therapy they need for better survival.

Olfactory receptor subgenome and expression in a highly olfactory procellariiform seabird.

Procellariiform seabirds rely on their sense of smell for foraging and homing. Both genomes and transcriptomes yield important clues about how olfactory receptor (OR) subgenomes are shaped by natural and sexual selection, yet no transcriptomes have been made of any olfactory epithelium of any bird species thus far. Here, we assembled a high-quality genome and nasal epithelium transcriptome of the Leach's storm-petrel (Oceanodroma leucorhoa) to extensively characterize their OR repertoire. Using a depth-of-coverage-assisted counting method, we estimated over 160 intact OR genes (∼500 including OR fragments). This method reveals the highest number of intact OR genes and the lowest proportion of pseudogenes compared to other waterbirds studied, and suggests that rates of OR gene duplication vary between major clades of birds, with particularly high rates in passerines. OR expression patterns reveal two OR genes (OR6-6 and OR5-11) highly expressed in adults, and four OR genes (OR14-14, OR14-12, OR10-2, and OR14-9) differentially expressed between age classes of storm-petrels. All four genes differentially expressed between age classes were more highly expressed in chicks compared to adults, suggesting that OR genes may exhibit ontogenetic specializations. Three highly differentially expressed OR genes also had high copy number ratios, suggesting that expression variation may be linked to copy number in the genome. We provide better estimates of OR gene number by using a copy number-assisted counting method, and document ontogenetic changes in OR gene expression that may be linked to olfactory specialization. These results provide valuable insight into the expression, development, and macroevolution of olfaction in seabirds.

Detecting copy number variation in next generation sequencing data from diagnostic gene panels

BackgroundDetection of copy number variation (CNV) in genes associated with disease is important in genetic diagnostics, and next generation sequencing (NGS) technology provides data that can be used for CNV detection. However, CNV detection based on NGS data is in general not often used in diagnostic labs as the data analysis is challenging, especially with data from targeted gene panels. Wet lab methods like MLPA (MRC Holland) are widely used, but are expensive, time consuming and have gene-specific limitations. Our aim has been to develop a bioinformatic tool for CNV detection from NGS data in medical genetic diagnostic samples.ResultsOur computational pipeline for detection of CNVs in NGS data from targeted gene panels utilizes coverage depth of the captured regions and calculates a copy number ratio score for each region. This is computed by comparing the mean coverage of the sample with the mean coverage of the same region in other samples, defined as a pool. The pipeline selects pools for comparison dynamically from previously sequenced samples, using the pool with an average coverage depth that is nearest to the one of the samples. A sliding window-based approach is used to analyze each region, where length of sliding window and sliding distance can be chosen dynamically to increase or decrease the resolution. This helps in detecting CNVs in small or partial exons. With this pipeline we have correctly identified the CNVs in 36 positive control samples, with sensitivity of 100% and specificity of 91%. We have detected whole gene level deletion/duplication, single/multi exonic level deletion/duplication, partial exonic deletion and mosaic deletion. Since its implementation in mid-2018 it has proven its diagnostic value with more than 45 CNV findings in routine tests.ConclusionsWith this pipeline as part of our diagnostic practices it is now possible to detect partial, single or multi-exonic, and intragenic CNVs in all genes in our target panel. This has helped our diagnostic lab to expand the portfolio of genes where we offer CNV detection, which previously was limited by the availability of MLPA kits.

Effects of Slide Storage on Detection of Molecular Markers by IHC and FISH in Endometrial Cancer Tissues From a Clinical Trial: An NRG Oncology/GOG Pilot Study.

We performed a pilot study in anticipation of using long-aged precut formalin-fixed paraffin-embedded tissue sections stored in real-world conditions for translational biomarker studies of topoisomerase 2A (TOP2A), Ki67, and human epidermal growth factor receptor 2 (HER2) in endometrial cancer. Formalin-fixed paraffin-embedded tissue blocks or unstained slides or both from GOG-0177 were collected centrally (1999-2000) and stored at room temperature. During 2004 to 2011 specimens were stored at 4°C. Matched pairs of stored slides and freshly cut slides from stored blocks were analyzed for TOP2A (KiS1), Ki67 (MIB1), and HER2 (HercepTest) proteins. To assess DNA stability (HER2 PathVision), fluorescence in situ hybridization (FISH) was repeated on stored slides from 21 cases previously shown to be HER2 amplified. Immunohistochemistry (IHC) staining intensity and extent, mean FISH copies/cell, and copy number ratios were compared using the κ statistic for concordance or signed rank test for differences in old cut versus new cut slides. IHC results reflected some protein degradation in stored slides. The proportion of cells with TOP2A staining was lower on average by 12% in older sections (P=0.03). The proportion of Ki67-positive cells was lower in stored slides by an average of 10% (P<0.01). Too few cases in the IHC cohort were FISH positive for any conclusions. HER2 amplification by FISH was unaffected by slide storage. We conclude that use of aged stored slides for proliferation markers TOP2A and Ki67 is feasible but may modestly underestimate true values in endometrial cancer. Pilot studies for particular storage conditions/durations/antigens to be used in translational studies are warranted.

Mixed ductal-lobular carcinoma: an analysis of CDH1 DNA copy number variation and mutation.

Mixed ductal-lobular carcinoma (MDL) of the breast is poorly understood. Dysfunction of E-cadherin, a cell adhesion protein encoded by the CDH1 gene located on 16q22.1, causes loss of cell adhesion and cellular polarity in lobular carcinoma (LC). This study focuses the aberrations of CDH1 in LC, ductal carcinoma (DC), and MDL to investigate the pathogenesis of MDL. The CDH1 DNA value (ratio of CDH1 copy number to the reference gene, RNase P) was calculated by digital polymerase chain reaction analysis of a total of 113 breast carcinoma cases (51 LCs, 54 DCs, and 8 MDLs). CDH1 gene mutation assay was performed for 20/51 LCs, 8/54 DCs, and 8 MDLs cases. The CDH1 DNA values were lower in LCs (average: 0.664) than in DCs (average: 1.296) (p < 0.000). In MDL, The CDH1 DNA values were significantly lower in LC areas (average: 0.58), compared to that of DC areas (average: 1.08) (p = 0.004), and there is no significant difference between the intermingled areas (average: 1.05) and DC areas (p = 0.775). Moreover, CDH1 mutations occurred more frequently in MDLs than in pure LCs and DCs. In one MDL case, the identical CDH1 mutation was found in LC and DC areas. Our study presented that MDL had more frequent CDH1 mutations. There were two possible processes for cancer cells in LC areas: one process was via DC areas with a common ancestor, and another was an independent process from DC areas.

The Acute Effects of Exercise and Temperature on Regional mtDNA.

A reduced mitochondrial DNA (mtDNA) copy number, the ratio of mitochondrial DNA to genomic DNA (mtDNA:gDNA), has been linked with dysfunctional mitochondria. Exercise can acutely induce mtDNA damage manifested as a reduced copy number. However, the influence of a paired (exercise and temperature) intervention on regional mtDNA (MINor Arc and MAJor Arc) are unknown. Thus, the purpose of this study was to determine the acute effects of exercise in cold (7 °C), room temperature (20 °C), and hot (33 °C) ambient temperatures, on regional mitochondrial copy number (MINcn and MAJcn). Thirty-four participants (24.4 ± 5.1 yrs, 87.1 ± 22.1 kg, 22.3 ± 8.5 %BF, and 3.20 ± 0.59 L·min−1 VO2peak) cycled for 1 h (261.1 ± 22.1 W) in either 7 °C, 20 °C, or 33 °C ambient conditions. Muscle biopsy samples were collected from the vastus lateralis to determine mtDNA regional copy numbers via RT-qPCR. mtDNA is sensitive to the stressors of exercise post-exercise (MIN fold change, −1.50 ± 0.11; MAJ fold change, −1.70 ± 0.12) and 4-h post-exercise (MIN fold change, −0.82 ± 0.13; MAJ fold change, −1.54 ± 0.11). The MAJ Arc seems to be more sensitive to heat, showing a temperature-trend (p = 0.056) for a reduced regional copy number ratio after exercise in the heat (fold change −2.81 ± 0.11; p = 0.019). These results expand upon our current knowledge of the influence of temperature and exercise on the acute remodeling of regional mtDNA.

Development and assessment of a duplex droplet digital PCR method for quantification of GM rice Kemingdao.

The implementation of genetically modified organism (GMO) labeling policies requires accurate quantitative methods to measure the GMO content in test samples. A Kemingdao/phospholipase D (KMD/PLD) duplex ddPCR method was established with rice genomic DNA (gDNA) of homozygous KMD as template by optimizing the annealing temperature and cycle number. Duplex ddPCR showed a linear response over the dynamic range from 68 to 175,000 copies, covering four orders of magnitude. The limit of detection (LOD) and limit of quantification (LOQ) for duplex ddPCR were determined to be 9 copies and 34 copies of the rice haploid genome, respectively. A very high dilution factor would result in unacceptable bias and coefficients of variation for determining copy number of the gDNA solution, and more than 1000 copies of the DNA template in one reaction is preferred to obtain accurate quantitative results by duplex PCR. Five blinded DNA samples with copy number ratio of 10%, 5%, 1%, 0.1%, and 0.05%, and three blinded real-life matrix samples with mass fraction of 5%, 1%, and 0.5% were quantified by duplex ddPCR, simplex ddPCR, and qPCR. These three methods all gave comparable GMO content and copy numbers within the required precision, but the duplex ddPCR showed the narrowest uncertainty interval and provided the highest precision in comparison to simplex ddPCR and qPCR. The ddPCR is a more appealing and reliable technology for the accurate quantification of GMO content than simplex ddPCR and qPCR considering the uncertainty and precision of quantitative results, the time consumption of generating droplets, and the cost of ddPCR reagents.

Maternal overweight but not paternal overweight before pregnancy is associated with shorter newborn telomere length: evidence from Guangxi Zhuang birth cohort in China

BackgroundTelomere length (TL) is variable at birth and is inversely associated with body mass index (BMI) in adulthood. A growing number of evidences suggested that a higher maternal pre-pregnancy BMI results in adverse offspring health outcomes, especially shorter newborn TL. However, a newborn’s genetic endowment is equally derived from both parents, the association between parental pre-pregnancy BMI and newborn TL has been rarely discussed. We aimed to determine the association between parental pre-pregnancy BMI and newborn TL.MethodsA total of 1082 parent-newborn pairs were recruited from the Guangxi Zhuang Birth Cohort (GZBC). TL in cord blood was measured using quantitative real-time polymerase chain reaction (qPCR) and expressed as the ratio of telomere copy number to single-copy gene number (T/S). A series of linear regressions were performed to assess the associations between parental pre-pregnancy BMI and newborn TL.ResultsMothers who were overweight before pregnancy had significantly shorter cord blood telomere length in their newborns than those who were normal weight before pregnancy [percentage change: − 7.96% (95% CI: − 14.49 to − 0.69%; P = 0.032)]. Further analysis of the combined effects of parental weight status on newborn TL showed that TL was significantly shortened among newborns whose mothers were overweight and fathers were of healthy weight when compared with those whose mothers and fathers were both of normal weight [percentage change: − 8.38% (95% CI: − 15.47 to − 0.92%; P = 0.028)]. Subgroup analysis indicated these effects were more pronounced among male newborns and those whose paternal age < 31 years or maternal age ≥ 28 years at delivery.ConclusionsMaternal pre-pregnancy overweight, but not paternal pre-pregnancy overweight is associated with shorter newborn TL. Weight control in reproductive women and effective healthy weight management before pregnancy may be of particular benefit for improving longevity and life quality of offspring.

The effect of ethanol concentration on the morphological and molecular preservation of insects for biodiversity studies.

Traditionally, insects collected for scientific purposes have been dried and pinned, or preserved in 70% ethanol. Both methods preserve taxonomically informative exoskeletal structures well but are suboptimal for preserving DNA for molecular biology. Highly concentrated ethanol (95–100%), preferred as a DNA preservative, has generally been assumed to make specimens brittle and prone to breaking. However, systematic studies on the correlation between ethanol concentration and specimen preservation are lacking. Here, we tested how preservative ethanol concentration in combination with different sample handling regimes affect the integrity of seven insect species representing four orders, and differing substantially in the level of sclerotization. After preservation and treatments (various levels of disturbance), we counted the number of appendages (legs, wings, antennae, or heads) that each specimen had lost. Additionally, we assessed the preservation of DNA after long-term storage by comparing the ratio of PCR amplicon copy numbers to an added artificial standard. We found that high ethanol concentrations indeed induce brittleness in insects. However, the magnitude and nature of the effect varied strikingly among species. In general, ethanol concentrations at or above 90% made the insects more brittle, but for species with robust, thicker exoskeletons, this did not translate to an increased loss of appendages. Neither freezing the samples nor drying the insects after immersion in ethanol had a negative effect on the retention of appendages. However, the morphology of the insects was severely damaged if they were allowed to dry. We also found that DNA preserves less well at lower ethanol concentrations when stored at room temperature for an extended period. However, the magnitude of the effect varies among species; the concentrations at which the number of COI amplicon copies relative to the standard was significantly decreased compared to 95% ethanol ranged from 90% to as low as 50%. While higher ethanol concentrations positively affect long-term DNA preservation, there is a clear trade-off between preserving insects for morphological examination and genetic analysis. The optimal ethanol concentration for the latter is detrimental for the former, and vice versa. These trade-offs need to be considered in large insect biodiversity surveys and other projects aiming to combine molecular work with traditional morphology-based characterization of collected specimens.

Association between nutrient intake and telomere length in Japanese female university students

Objective Telomere length can be a biomarker of cumulative oxidative stress and inflammation indicating biological aging. Previous studies examined association of nutrient intake with telomere length targeting middle-aged and elderly individuals. This study examined whether dietary macro- and micronutrient intake was associated with telomere length in young females. Methods Seventy-four Japanese young females (median (interquartile range) age was 19 (19 − 20) years) participated. We estimated their intake of nutrients (energy, protein, fat, carbohydrate, essential elements, vitamins, fatty acids, and dietary fibre) using a semi-quantitative food frequency questionnaire and measured telomere length (T/S ratio, the ratio of telomere repeat copy number (T) to single-copy gene number (S)) of DNA extracted from blood by qPCR. The association between telomere length and tertiles of nutrient intake were analysed. Results The median (interquartile range) of telomere length was 0.70 (0.52 − 0.98). Vitamin A intake was positively associated with telomere length (tertile 1 vs. 2, coefficient [95% confidence interval] = 0.42 [0.12, 0.71]; tertile 1 vs. 3, coefficient [95% confidence interval] = 0.33 [0.04, 0.62]) after adjusting for covariates (age, BMI, passive smoking, and drinking). Conclusions Our findings suggest that variation in vitamin A intake might influence telomere attrition in healthy individuals.